Refined RIP-seq protocol for epitranscriptome analysis with low input materials.

Area of Science:

• Molecular Biology
• Epigenetics
• Cancer Research

Background:

• N6-Methyladenosine (m6A) is the most abundant internal mRNA modification in mammals.
• m6A RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq) maps m6A sites but requires substantial RNA input.
• Current MeRIP-seq protocols are unsuitable for limited clinical samples like patient tumors.

Purpose of the Study:

• To refine the m6A MeRIP-seq protocol for low-input RNA samples.
• To enable m6A epitranscriptome profiling in patient tumors.
• To investigate m6A dynamics correlating mRNA and protein levels in lung adenocarcinoma.

Main Methods:

• Optimization of m6A MeRIP-seq parameters including RNA input, fragmentation, antibody selection, and washing/elution conditions.
• Development of a post-amplification rRNA depletion strategy for low-input samples.
• Bioinformatic analysis pipeline for m6A peak identification and integration with transcriptome and proteome data.

Main Results:

• A refined m6A MeRIP-seq protocol successfully profiled the epitranscriptome from 500 ng of total RNA.
• Approximately 12,000 high signal-to-noise m6A peaks were identified in lung adenocarcinoma patient tumors.
• Integrative analysis revealed m6A-driven dynamics explaining mRNA-protein level discordance.

Conclusions:

• The optimized low-input m6A MeRIP-seq method is effective for clinical sample analysis.
• This technique facilitates the study of m6A epitranscriptome dynamics in patient tumors.
• The findings provide a foundation for understanding m6A mechanisms in clinical settings.